Project Summary/Abstract The overall goal of this research is to understand how one protein sequence can interconvert between two stable, unrelated native state structures. Like other chemokines, human lymphotactin (Ltn/XCL1) induces chemotactic cell migration but structural characterization revealed a uniquely metamorphic native state. In the last grant period we showed that Ltn undergoes a reversible rearrangement that completely disrupts every stabilizing interaction. We solved the structure of each species by adjusting solution conditions (temperature and ionic strength) to shift the conformational equilibrium from one extreme to the other. One native state (Ltn10) corresponds to the conserved chemokine fold and activates XCR1, the specific G protein-coupled receptor for lymphotactin. Rearrangement to the other native conformation (Ltn40) produces a novel fourstranded sheet that dimerizes to form a beta sandwich with high affinity for cell-surface glycosaminoglycans (GAG). Ltn10-Ltn40 interconversion is essential for lymphotactin to function as a chemoattractant in vivo, but the forces stabilizing each state and the pathway linking them remain unknown. To identify unique structural and dynamic features that stabilize each of the Ltn native-state conformations (aim 1), we will characterize backbone dynamics from NMR relaxation measurements, perform long (>100 ns) atomistic MD simulations in conditions (temperature and ionic strength) that favor each of the dual Ltn native state structures, and measure the influence of specific mutations on the conformational equilibrium. To demonstrate that the essential contacts required for the dual native state have been defined, metamorphic folding will be introduced into a different member of the chemokine family. In aim 2, we will combine kinetic measurements on the &#956;s-s timescales from NMR and fluorescence to characterize the structural transition between Ltn10 and Ltn40 and establish the mechanistic relationship between interconversion and folding. Aim 3 will test the hypothesis that small molecule ligands can function as allosteric inhibitors by altering the Ltn conformational equilibrium. GAG binding favors the Ltn40 species, and we predict that the XCR1 N-terminus will shift the conformational equilibrium by recognizing an epitope unique to Ltn10. We will define this chemokine-receptor interface by NMR, and exploit the sulfotyrosine recognition site for structure-based discovery of novel ligands that should also drive the interconversion reaction toward Ltn10. Finally, we will investigate the hypothesis that a direct interaction between the unique Ltn C-terminal domain and CD4 is required for Ltn-mediated apoptosis of CD4+ T cells.